On-Target Action of Anti-Tropomyosin Drugs Regulates Glucose Metabolism

www.nature.com/scientificreports OPEN On-target action of anti- tropomyosin drugs regulates glucose metabolism Received: 30 November 2017 Anthony J. Kee1, Jayshan Chagan1, Jeng Yie Chan2, Nicole S. Bryce 1, Christine A. Lucas1, Accepted: 1 March 2018 Jun Zeng3, Jef Hook1, Herbert Treutlein 4, D. Ross Laybutt2, Justine R. Stehn1,5, Published: xx xx xxxx Peter W. Gunning1 & Edna C. Hardeman1 The development of novel small molecule inhibitors of the cancer-associated tropomyosin 3.1 (Tpm3.1) provides the ability to examine the metabolic function of specifc actin flament populations. We have determined the ability of these anti-Tpm (ATM) compounds to regulate glucose metabolism in mice. Acute treatment (1 h) of wild-type (WT) mice with the compounds (TR100 and ATM1001) led to a decrease in glucose clearance due mainly to suppression of glucose-stimulated insulin secretion (GSIS) from the pancreatic islets. The impact of the drugs on GSIS was signifcantly less in Tpm3.1 knock out (KO) mice indicating that the drug action is on-target. Experiments in MIN6 β-cells indicated that the inhibition of GSIS by the drugs was due to disruption to the cortical actin cytoskeleton. The impact of the drugs on insulin-stimulated glucose uptake (ISGU) was also examined in skeletal muscle ex vivo. In the absence of drug, ISGU was decreased in KO compared to WT muscle, confrming a role of Tpm3.1 in glucose uptake. Both compounds suppressed ISGU in WT muscle, but in the KO muscle there was little impact of the drugs. Collectively, this data indicates that the ATM drugs afect glucose metabolism in vivo by inhibiting Tpm3.1’s function with few of-target efects. As well as forming part of the striated muscle contractile apparatus, the actin cytoskeleton is involved in many fundamental processes in all eukaryotic cells including cell proliferation, cell migration, cell adhesion and intra- cellular trafcking. Te high sequence homology between the 6 actin isoforms1 has made it difcult to design compounds that discriminate between the diferent actin flaments in cells based on their actin composition2,3. Te discovery that the tropomyosins (Tpms), which form co-polymers with actin, defne the intrinsic func- tional capacity of the flament has provided the ability to discriminate between diferent flament populations4. Tpms are encoded by four genes and many isoforms (n > 40) arise from these genes by exon splicing and alter- nate promoter usage1. Tree isoforms are striated muscle-specifc and form part of the actin thin flament of the contractile apparatus where they regulate actin-myosin interactions and give strength and stability to the contractile apparatus5. Te other Tpm isoforms are considered to be non-muscle or cytoskeletal. Previous studies have demonstrated that both Tpm and actin isoforms segregate into functionally distinct flament populations in diferent cell types6–16. Tpms are now viewed as the ‘gate keepers of the actin cytoskeleton’17, regulating the interaction of other actin regulatory proteins with the actin flament. Tis provides a means to independently regulate the cytoskeleton at diferent sites within the cell and to tailor the function of actin flaments at these diferent sites. Tis also provides the opportunity to target distinct actin flament populations in cells based on Tpm composition. Studies using gene knock-out and overexpressing transgenic approaches have demonstrated that Tpms in an isoform-specifc manner regulate a number of specifc physiological processes in yeast, insect and mammalian systems4,18,19. Tpm3.1 has been shown to be essential for embryonic stem cell proliferation20, to regulate organ size and cell proliferation in mice10 and is required for cancer cell survival21. Cell transformation is accompanied by changes in the Tpm isoform composition of their actin cytoskeleton4 with Tpm3.1 and Tpm4.2 consistently retained by all human cancer cells thus far examined21. Tpm4.2 has also been shown to have an important role 1School of Medical Sciences, UNSW Sydney, Sydney, NSW, 2052, Australia. 2Garvan Institute of Medical Research, St Vincent’s Hospital, UNSW Sydney, Sydney, NSW, Australia. 3MedChemSoft Solutions, Level 3 Brandon Park Drive, Wheelers Hill, 3150, VIC, Australia. 4Sanoosa Pty. Ltd., 35 Collins Street, Melbourne, 3000, VIC, Australia. 5Novogen Pty Ltd, 502/20 George St, Hornsby, NSW, 2077, Australia. Correspondence and requests for materials should be addressed to E.C.H. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:4604 | DOI:10.1038/s41598-018-22946-x 1 www.nature.com/scientificreports/ in the terminal stages of platelet production22. In two recent studies, Tpm3.1-containing actin flaments have also been shown to regulate glucose uptake in mice in an isoform-specifc manner23,24. In the frst study, insulin stimulation of Akt resulted in phosphorylation of Tmod3, capping Tpm3.1-containing actin flaments that are involved in actin reorganization at the cell periphery and increased incorporation of the GLUT4 glucose trans- porter into the plasma membrane (PM)24. In the other study, Tpm3.1 was shown to regulate insulin-stimulated glucose uptake in mouse skeletal muscle and adipose tissue23. Evidence was also provided for two populations of actin flaments required for GLUT4 vesicle trafcking, one containing Tpm3.1 and MyoIIA motors, and the other lacking Tpm, but interacting with Myo1c23. We have developed compounds that target Tpm3.1 as anti-cancer agents21,25. Tese anti-Tpm (ATM) compounds, disrupt Tpm3.1-containing actin flaments in cancer and non-cancer cells21,23,25,26, are anti-proliferative and have anti-tumor activity in mouse xenograph models21,25. In vitro experiments using purifed proteins showed that these compounds disrupt Tpm3.1-regulated actin flament dynamics by increasing the rate of flament depo- lymerisation21,25,26. In the present study, we show that the ATM compounds impacted two processes known to be regulated by the actin cytoskeleton, insulin-stimulated glucose uptake and insulin secretion27,28. Comparison of the efect of the compounds in wild-type (WT) versus Tpm3.1 knock-out (KO) mice indicate that the compounds are operating by specifcally inhibiting Tpm3.1 function. Results Strategy to assess on- and of-target impact of the ATM compounds. Te major objective of this study was to establish if ATM drugs can specifcally target the role of Tpm3.1 in glucose metabolism as we had previously shown that a major phenotype in Tpm3.1 transgenic and knock-out (KO) mice was altered glucose uptake23. Te on- and of-target impact of the compounds were evaluated by comparing the impact of the drugs in wild-type versus Tpm3.1 KO mice. Confrmation of an on-target efect was shown if there was no impact of the drug in KO mice, and the response to the drug in the WT mice mimicked the vehicle-treated KO mice. ATM-1001 structure and impact on Tpm3.1 flaments. Te impact of two ATM compounds was examined, our first-in-class compound, TR10021 and a novel compound, ATM-1001 which was identified from a previously described library of compounds on the basis of its cytotoxic potency and ability to disrupt Tpm3.1-containing microflaments25. Molecular modelling of ATM1001 with a peptide containing the last 37 amino acids of Tpm3.1 shows that the scafold of ATM-1001 fts into the cavity between two helixes of Tpm showing close contacts with Leu31 and Met37 of the C-terminal peptide (corresponding to Leu243 and Met248 in the full-length Tpm3.1 protein) (Fig. 1A). Te trimethylammonium [N-Me3(+)] charged group of ATM1001 interacts with the Tpm C-terminus through electrostatic interactions, while the phenyl ring provides hydro- phobic interactions with Leu27. Using a High Content Imaging (HCI) assay, we found that Tpm3.1-containing flament bundles in MEF cells were disrupted by 5 μM ATM-1001 (Fig. 1B) and showed that ATM-1001 inhibits Tpm3.1’s ability to protect actin from depolymerization by quantifying polymerized actin over time (Fig. 1C,D). These activities are similar to that reported for other anti-tropomyosin (ATM) compounds (TR100 and ATM-3507)21,25,26. ATM-1001 plasma clearance and tissue distribution. We examined the plasma clearance and distribution of ATM-1001 into metabolically active tissues following a single bolus injection of the compound (40 mg/ kg body weight) in wild-type and Tpm3.1 KO mice. ATM-1001 was cleared rapidly from the blood and reached a maximum concentration in the tissues (skeletal muscle and pancreas) at 1–2 h afer injection (Figure S1). Interestingly, the drug concentration in pancreas was higher than muscle, perhaps refecting a greater blood fow to the visceral tissues compared to muscle. Importantly, the plasma clearance and tissue distribution of ATM- 1001 were similar in WT and KO mice (Figure S1) indicating that Tpm3.1 is not involved in drug clearance and tissue distribution. ATM drugs alter glucose clearance via impacts on Tpm3.1. To assess the impact of the ATM compounds on glucose metabolism we frst confrmed that injection of the vehicle alone had no impact on glucose clearance compared to saline-injected controls (Figure S2A). To provide an initial assessment of the impact of the ATM compounds on glucose metabolism, ATM drugs (40 mg/kg BW, i.p.) and vehicle (Dexolve-7) were admin- istered to the Tpm3.1 WT and KO mice and 1 h later an intraperitoneal glucose tolerance test was performed (see Fig. 2A for study design). One hour following TR100 or ATM-1001 injection blood glucose was similar to vehicle-injected controls (Figure S3A,C). However, both TR100 and ATM-1001 signifcantly decreased glucose clearance in WT and KO mice (Fig. 2B–E). Importantly, the impact of the drugs on clearance was signifcantly less in the KO versus the WT mice for both TR100 and ATM-1001 (Fig.

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